US20210164024A1 - Method for detecting nucleic acid based on prokaryotic argonaute protein and application thereof - Google Patents

Method for detecting nucleic acid based on prokaryotic argonaute protein and application thereof Download PDF

Info

Publication number
US20210164024A1
US20210164024A1 US17/045,393 US201817045393A US2021164024A1 US 20210164024 A1 US20210164024 A1 US 20210164024A1 US 201817045393 A US201817045393 A US 201817045393A US 2021164024 A1 US2021164024 A1 US 2021164024A1
Authority
US
United States
Prior art keywords
nucleic acid
detection system
target nucleic
acid molecules
amplification
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
US17/045,393
Other languages
English (en)
Inventor
Yan Feng
Guanhua XUN
Qian Liu
Yuesheng CHONG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiaohong Biotechnology Shanghai Co Ltd
Original Assignee
Shanghai Jiaotong University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Jiaotong University filed Critical Shanghai Jiaotong University
Assigned to SHANGHAI JIAO TONG UNIVERSITY reassignment SHANGHAI JIAO TONG UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FENG, YAN, LIU, QIAN, CHONG, Yuesheng, XUN, Guanhua
Publication of US20210164024A1 publication Critical patent/US20210164024A1/en
Assigned to FENG, YAN reassignment FENG, YAN ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SHANGHAI JIAO TONG UNIVERSITY
Assigned to JIAOHONG BIOTECHNOLOGY (SHANGHAI) CO., LTD. reassignment JIAOHONG BIOTECHNOLOGY (SHANGHAI) CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FENG, YAN
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

Definitions

  • the present invention is in the field of biotechnology, and in particular relates to a method for detecting nucleic acid based on prokaryotic Argonaute protein and application thereof.
  • Nucleic acid detection technology is widely used in many fields such as molecular medical diagnosis, food safety inspection and environmental monitoring. Rapid, cheap and sensitive nucleic acid detection can be widely used in pathogen detection, genotyping, and disease course monitoring.
  • nucleic acid detection methods using gene editing enzymes include the methods using C2C2 enzyme-mediated collateral cleavage effect to detect target RNA.
  • C2C2 enzyme-mediated collateral cleavage effect to detect target RNA.
  • target DNA especially trace DNA.
  • the purpose of the present invention is to provide a method based on nucleic acid detection with high sensitivity, good specificity and high throughput to target DNA, and the application thereof.
  • a detection system for detecting target nucleic acid molecules wherein the system comprises:
  • the target nucleic acid molecule is target DNA
  • the guide ssDNA pair includes a sense strand guide ssDNA and an antisense strand guide ssDNA.
  • the guide ssDNA is a 5′-phosphorylated single-stranded DNA molecule.
  • the guide ssDNA has a length of n bases, and n ⁇ 14.
  • the length of the guide ssDNA is 14-60 nt, preferably 16-40 nt.
  • the number of the guide ssDNAs is one or more pairs.
  • the PfAgo enzyme is derived from the archaea Pyrococcus furiosus.
  • the PfAgo includes wild type PfAgo and mutant PfAgo.
  • the mutation sites corresponding to different types of the target nucleic acid molecules are at positions 10 and 11 of the guide ssDNA.
  • the detection system further comprises (d) a buffer.
  • the detection system further comprises primers for amplifying the target nucleic acid molecules.
  • the detection system further comprises the target nucleic acid molecules to be detected.
  • the target nucleic acid molecules (or the amplification product thereof) are cleaved by the PfAgo enzyme to produce a secondary guide ssDNA.
  • sequences of the secondary guide ssDNA and the fluorescent reporter nucleic acid are complementary.
  • the PfAgo enzyme is guided to cleave the fluorescent reporter nucleic acid, thereby generating a detectable signal (such as fluorescence).
  • the concentration of the target nucleic acid molecules to be detected in the detection system is 1-1000 copies/microliter or 10 3 -10 10 copies/microliter, preferably 100-1000 copies/microliter, more preferably 1-100 copies/microliter.
  • the concentration of the target nucleic acid molecules to be detected in the detection system is 1 fM-200 pM, preferably 1-1000 fM, more preferably 1-100 fM, and most preferably 1-20 fM.
  • the working temperature of the gene editing enzyme is 87-99° C.
  • the concentration of the fluorescent reporter nucleic acid is 100-1000 nM.
  • the molar ratio of the fluorescent reporter nucleic acid to the target nucleic acid molecules is 10 3 :1 to 10 8 :1, preferably 10 4 :1 to 10 7 :1.
  • the target DNA comprises cDNA.
  • the target DNA is selected from the group consisting of: single-stranded DNA (including cDNA), double-stranded DNA, and a combination thereof.
  • the fluorescent group and the quenching group are independently located at the 5′ end and the 3′ end of the fluorescent reporter nucleic acid.
  • the length of the fluorescent reporter nucleic acid is 9-100 nt, preferably 10-60 nt, more preferably 15-40 nt.
  • the target nucleic acid molecules comprise target nucleic acid molecules derived from a species selected from the group consisting of: a plant, an animal, a microorganism, a virus, and a combination thereof.
  • the target DNA is a synthetic or a naturally occurring DNA.
  • the target DNA comprises a wild-type or a mutant DNA.
  • kit for detecting target nucleic acid molecules comprising:
  • the kit may further comprises a buffer.
  • the kit comprises:
  • the kit further comprises:
  • the buffer for enzyme digestion comprises MnCl 2 .
  • the kit further comprises:
  • a third aspect of the invention provides a method for detecting the presence or absence of target nucleic acid molecules in a sample, comprising the steps of:
  • the fluorescence signal value is detected in the first reaction solution, it indicates that there is target nucleic acid molecules in the sample; and if the fluorescence signal value is not detected in the first reaction solution, it indicates that there is no target nucleic acid molecule in the sample.
  • the sample to be detected comprises an unamplified sample and an amplified (or nucleic acid amplified) sample.
  • the sample to be detected is a sample obtained by amplification.
  • the nucleic acid amplification method is selected from the group consisting of: PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification and rolling circle amplification.
  • the PCR comprises high temperature PCR, normal temperature PCR, and low temperature PCR.
  • the method is for detecting whether there is an SNP, a point mutation, a deletion, and/or an insertion for nucleic acids at a target site.
  • a microplate reader or a fluorescence spectrophotometer is used in the fluorescence detection.
  • the method is an in vitro method.
  • the method is non-diagnostic and non-therapeutic.
  • nuclease Pyrococcus furiosus Argonaute for preparing reagents or kits for detecting target nucleic acid molecules based on secondary cleavage.
  • the enzyme Pyrococcus furiosus Argonaute is derived from the archaea Pyrococcus furiosus ; or the homologous analogue thereof with the same or similar functions.
  • the PfAgo includes wild type PfAgo and mutant PfAgo.
  • FIG. 1 is a schematic diagram showing the first cleavage of the gene-encoding enzyme of the present invention, in which a pair of guide ssDNAs is designed for the same DNA strand, and the corresponding cleavage sites are marked by black arrows, and a blunt end is formed after the action of pfAgo enzyme.
  • FIG. 2 is a schematic diagram showing the second cleavage of the gene-encoding enzyme of the present invention, in which the specific cleavage guided by the primary guide ssDNAs produces the secondary guide ssDNAs.
  • FIG. 3 shows the steps and principles of the detection method of the present invention.
  • FIG. 4 shows the specificity and sensitivity of the nucleic acid detection system.
  • FIG. 5 shows the results of the multiplex experiment of the nucleic acid detection system in an example of the present invention.
  • the CON1-1b-FAM reporter nucleic acid molecule no template was used;
  • JFH-1 2a-VIC reporter nucleic acid molecule JFH-1 2a template
  • FIG. 6 shows the multiple detection result of different combinations of HPV, using plasmids as the samples.
  • FIG. 7 shows the multiple detection result of high-risk subtype HPV16/HPV18, in the clinical samples.
  • the method of the present invention utilizes the characteristics of the PfpAgo enzyme, that is, after the first cleavage mediated by the primary guide ssDNA (guide ssDNA), at a suitable reaction temperature (such as about 90-98 degrees), the broken 5′ nucleic acid fragments can be re-used by the PfAgo enzyme for the cutting of the complementary fluorescent reporter nucleic acid strand thereof.
  • a suitable reaction temperature such as about 90-98 degrees
  • the terms “the detection system of the present invention” and “the nucleic acid detection system based on Argonaute protein” are used interchangeably and refer to the detection system described in the first aspect of the present invention.
  • the terms “the detection method of the present invention” and “the nucleic acid detection method based on Argonaute protein” are used interchangeably and refer to the detection method described in the second aspect of the present invention.
  • the terms “gene editing enzyme Pyrococcus furiosus ”, “nuclease Pyrococcus furiosus ”, and “PfAgo enzyme” are used interchangeably and refer to the enzyme described in the first aspect of the present invention.
  • the term “secondary cleavage” means that in the detection method of the present invention, in the presence of the primary guide ssDNAs, the Ago enzyme of the present invention cuts the target nucleic acid sequence to form a new 5′ phosphorylated nucleic acid sequence (secondary guide ssDNA); then, in the presence of the PfAgo enzyme, the secondary guide ssDNA continues to guide the PfAgo enzyme to cleave the fluorescent reporter nucleic acid complementary to the secondary guide ssDNA.
  • This mode of specific cleavage for the target nucleic acid sequence first (first cleavage), and then specific cleavage for the fluorescent reporter nucleic acid (second cleavage), is defined as a “secondary cleavage”. In the present invention, both the first cleavage and the second cleavage are specific.
  • a core component is a gene editing enzyme, such as Ago enzyme.
  • a preferred Ago enzyme is PfAgo enzyme, which is derived from the archaea Pyrococcus furiosus , with a gene length of 2313 bp and an amino acid sequence consisting of 770 amino acids.
  • the cleavage characteristics of PfAgo enzyme are: the enzyme can use 5′ phosphorylated oligonucleotide as the guide ssDNA to guide the enzyme to precisely cleave the target nucleic acid sequence; and the cleavage site is located at the phosphodiester bond between the target nucleic acids (ssDNA) corresponding to the 10th and the 11th nucleotides of the guide ssDNA.
  • ssDNA target nucleic acids
  • the preferred working temperature of PfAgo enzyme is 95 ⁇ 2 degrees.
  • a core component is a guide ssDNA pair.
  • the preferred guide ssDNAs are all oligonucleotides with a length of 14-24 nt (e.g. 16 nt), and the first 5′ nucleotides are all phosphorylated thymine (T).
  • a pair of guide ssDNAs for the same DNA strand is binded to the target nucleic acid molecule, and the corresponding cleavage sites are marked by black arrows, and a blunt end is formed after the action of pfAgo enzyme.
  • a core component is a reporter nucleic acid carrying a reporter molecule.
  • the preferred reporter molecules are fluorescent molecules or fluorescent groups.
  • a preferred reporter nucleic acid molecule is a nucleic acid molecule that carries a fluorescent group and a quenching group, respectively.
  • a fluorescent group (F) is labeled at the 5′ end
  • a quenching group (Q) is labeled at the 3′ end.
  • FIG. 2 shows a fluorescent reporter nucleic acid with a length of 17 nt, a fluorescent group (F) at the 5′ end, and a quenching group (Q) at the 3′ end.
  • the fluorescent reporter nucleic acid is determined according to the produced position of the secondary guide ssDNA; the target nucleic acid sequence is cleaved by the primary guide ssDNA to form a new 5′ phosphorylated nucleic acid sequence, which is called the secondary guide ssDNA, and the fluorescent reporter nucleic acid covers the corresponding position of the secondary guide ssDNA (for example, 1st-16th bases).
  • the present invention provides a detection system for detecting target nucleic acid molecules, which comprises:
  • the target nucleic acid molecule is target DNA
  • the present invention also provides a nucleic acid detection method based on the gene editing enzyme Pyrococcus furiosus Argonaute (PfAgo).
  • a series of guide ssDNAs can be designed according to the different target nucleic acid sequences. These guide ssDNAs target the nucleic acid to be detected and mediate the PfAgo enzyme to cleave the target fragment, to form a new secondary guide ssDNA. In the presence of the PfAgo enzyme, the secondary guide ssDNA continues to guide the PfAgo enzyme to cleave the fluorescent reporter nucleic acid complementary to the secondary guide ssDNA, so as to achieve the detection of the target nucleic acid.
  • the PfAgo enzyme can selectively cleave nucleic acid sequences that have differences in some sites, thereby realizing typing detection.
  • the mutation sites corresponding to different types are placed in the 10th and 11th positions of the guide ssDNA. Due to the specificity of pfAgo enzyme selection, two consecutive point mutations can inhibit the cleavage activity, so as to achieve the detection of different types.
  • the present invention provides primers, guide ssDNAs, and fluorescent reporter nucleic acids for nucleic acid detection, for example, for the detection of the target gene PIK3CA E545K, or two types of HCV virus, JFH-1 2a and CON1-1b, respectively.
  • multiple target nucleic acids to be detected and guide ssDNAs can be added to the cleavage system of PfAgo enzyme at the same time, and the reporter nucleic acids with different fluorescent groups can be combined to achieve multiple detection of target nucleic acids.
  • the method of the present invention is very suitable for detecting trace amounts of nucleic acids.
  • the present invention can detect target nucleic acids with a concentration as low as fM level.
  • the detection method of the present invention comprises the following steps:
  • step 1 designing amplification primers, specific oligonucleotide guide ssDNAs and fluorescent reporter nucleic acids for different target nucleic acid sequences to be detected;
  • step 2 collecting samples to be tested and extracting nucleic acid complexes containing the target sequence
  • step 3 using the obtained sample to be tested as the templates and adding different amplification primer pairs to perform a pre-amplification reaction;
  • step 4 adding specific oligonucleotide guide ssDNAs, corresponding fluorescent reporter nucleic acids and PfAgo enzyme to the pre-amplification reaction system of step 3, to perform specific cleavage under the condition of continuous incubation at 95 degrees;
  • step 5 performing quantitative real-time PCR analysis on the system of step 4;
  • step 6 analyzing the image, and then adjusting the Start value, End value and threshold line of the Baseline to determine the result.
  • the Tm value of the amplification primer used in the amplification reaction is usually about 60 ⁇ 3 degrees, and the size of the amplified fragment is about 90-120 bp.
  • the amplification primer design should avoid the region to be detected.
  • the invention also provides a kit for the detection method of the invention.
  • the kit comprises:
  • the kit further comprises:
  • the buffer for gene editing enzyme digestion comprises MnCl 2 .
  • the kit further comprises:
  • the present invention is particularly suitable for the detection of trace target nucleic acid molecules and multiple detections, and has a wide applicability.
  • the target nucleic acid molecule may be DNA or RNA.
  • the target nucleic acid molecule is RNA, it can be converted into DNA by reverse transcription and then detected.
  • the target nucleic acid molecules comprise target nucleic acid molecules derived from a species selected from the group consisting of: a plant, an animal, a microorganism, a virus, and a combination thereof.
  • the target DNA is a synthetic or a naturally occurring DNA.
  • the target DNA comprises a wild-type or a mutant DNA.
  • the present invention can perform proactive management of diseases such as prediction and prevention, and achieve early detection and early treatment, or early prediction and early prevention. Due to the high detection sensitivity of the present invention, it is suitable for early diagnosis, thus for an antidote against the disease, saving the patient's treatment time and improving the treatment success rate. The invention reduces high medical cost and waste, and strives for a golden opportunity for treatment.
  • the present invention can accurately, conveniently and rapidly identify nucleic acid molecules in environmental pollutants and provide effective environmental detection data.
  • the nucleic acid detection method based on the gene editing enzyme Pyrococcus furiosus Argonaute (PfAgo) of the present invention fully utilizes the cleavage characteristics of the enzyme, making it a highly specific detection method.
  • a variety of target nucleic acids to be detected with the corresponding primary guide ssDNAs and fluorescent reporter nucleic acids can be added to the reaction system of the present invention at the same time, to realize single-tube multiple detection.
  • the nucleic acid detection method of the present invention has high sensitivity, and the detection limit of nucleic acid is aM-fM level.
  • the nucleic acid detection method of the present invention has good specificity and can distinguish different types of nucleic acid sequences.
  • the nucleic acid detection method of the present invention has convenient operation, simple design and low price.
  • the nucleic acid detection method of the present invention based on the gene editing enzyme Pyrococcus furiosus Argonaute (PfAgo) and the usage method thereof.
  • SEQ ID NO: 1 5′-CTGTGACTCCATAGAAAATCTTTCTCCTGCT CAGTGATTTCAGAGAGGATCTCGTGTAGAAAT TGCTTTGAGCTGTTCTTTGTCATTTTCCCT-3′.
  • the corresponding detection reagents include:
  • amplification primers F-primer and R-primer wherein the specific sequences are as follows:
  • F-primer (SEQ ID NO: 2) 5′-CTGTGACTCCATAGAAAATCTTTCTCC-3′
  • R-primer (SEQ ID NO: 3) 5′-AGGGAAAATGACAAAGAACAGCTC-3′
  • a specific guide ssDNA pair comprising a sense strand guide ssDNA and an antisense strand guide ssDNA, wherein the specific sequences are as follows:
  • sense strand guide ssDNA (SEQ ID NO: 4) 5′P-TTCTCCTGCTCAGTGA-3′ antisense strand guide ssDNA: (SEQ ID NO: 5) 5′P-TGAAATCACTGAGCAG-3′
  • Fluorescent reporter nucleic acid 5′ FAM (fluorescent group)-CTCGTCCTCTTTCTAAA-BHQ1 (quenching group) 3′ (SEQ ID NO: 6)
  • an enzyme preparation for amplification reaction such as AceQ qPCR Probe Master Mix (Vazyme)
  • FIG. 3 The schematic diagram of the nucleic acid detection method based on the gene editing enzyme Pyrococcus furiosus Argonaute (PfAgo) of the present invention is shown in FIG. 3 .
  • the specific operation steps are as follows:
  • the dry powders of the amplification primers F-primer and R-primer were dissolved in ultrapure water to prepare a 10 uM storage solution.
  • the dry powders of sense strand guide ssDNA and antisense strand guide ssDNA were dissolved in ultrapure water to prepare a 100 uM storage solution.
  • the fluorescent reporter nucleic acid dry powder was dissolved in ultrapure water to prepare a 10 uM storage solution.
  • the amplification system was put into a PCR machine for amplification reaction (pre-denaturation at 95 degrees for 5 min, denaturation at 95 degrees for 15 sec, extension at 60 degrees for 15 sec, 30 cycles).
  • step (4) After the amplification reaction was completed, PfAgo enzyme, MnCl 2 , sense strand guide ssDNA, antisense strand guide ssDNA, and fluorescent reporter nucleic acid were added to the system in step (4), to make it a 25 uL reaction system (the final concentration of PfAgo enzyme was 200 nM, and the final concentration of MnCl 2 was 500 uM, and the final concentration of guide ssDNAs was 2 uM, and the final concentration of fluorescent reporter nucleic acid was 400 nM).
  • step (5) The reaction system in step (5) was placed on a fluorescent quantitative PCR instrument for detection (incubation at 95 degrees for 30 min, and the fluorescent signal was detected once every minute).
  • the specific target nucleic acids (SEQ ID NO: 1) was diluted according to the principle of 10-fold dilution method, into standard stock solutions of 200 pM, 20 pM, 2 pM, 200 fM, 20 fM, 2 fM and 0 fM.
  • the nucleic acid standard stock solutions of different concentrations were added to the reaction system described in Example 1. The sample was added and the reaction was carried out according to the steps, and the fluorescent signal value at the wavelength of the corresponding fluorescent group was detected by fluorescent quantitative PCR.
  • FIGS. 4A-4H The results are shown in FIGS. 4A-4H .
  • the concentrations of the target nucleic acids in FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G were 0 fM, 2 fM, 20 fM, 200 fM, 2 pM, 20 pM, 200 pM, and
  • FIG. 4H shows the result of the non-target nucleic acids (20 ng was added to the system).
  • the non-target nucleic acid was the total DNA extracted from normal human serum. The results show that even if 20 ng of irrelevant non-target nucleic acids were added to the system, no positive results would be produced.
  • the method of the present invention can detect target nucleic acid molecules down to the fM level.
  • the method of the present invention has very high specificity.
  • Solutions of different types of target nucleic acids with a concentration of 200 pM were prepared, which were the two types of HCV viruses JFH-1 2a and CON1-1b, respectively.
  • FIG. 5 shows the specificity of the fluorescent reporter nucleic acids, which did not affect each other in one reaction system.
  • PfAgo enzyme MnCl 2 , 2 groups of specific ssDNAs, 2 pairs of fluorescent reporter nucleic acids with different fluorescent groups (JFH-1 2a type corresponded VIC fluorescence, CON1-1b type corresponded FAM fluorescence) were added to the same reaction tube, and the detection was carried out according to the reaction steps in Example 1.
  • FIGS. 5A-5F The results are shown in FIGS. 5A-5F .
  • the results show that when the target nucleic acid to be detected was a blank control, no fluorescence signal value was generated; when the target nucleic acid to be detected was JFH-1 2a, only VIC fluorescence was generated; when the target nucleic acid to be detected was CON1-1b, only FAM fluorescence was generated; when the target nucleic acids to be detected were JFH-1 2a and CON1-1b, both FAM and VIC fluorescences were generated simultaneously.
  • the nucleic acid detection method of the present invention can be used for multiple detection of a single tube.
  • HPV-6 SEQ ID NO: 19
  • HPV-11 SEQ ID NO: 20
  • HPV-16 SEQ ID NO: 21
  • HPV-18 SEQ ID NO: 22
  • PfAgo enzyme MnCl 2 , 4 kinds of specific ssDNAs, 4 kinds of fluorescent reporter nucleic acids with different fluorescent groups (HPV-6 type corresponded NED fluorescence, HPV-11 type corresponded ROX fluorescence, HPV-16 type corresponded FAM fluorescence, HPV-18 type corresponded JOE fluorescence) were added to the same reaction tube, and the detection was carried out according to the reaction steps in Example 1.
  • the result is shown in FIG. 6 .
  • the results show that when the target nucleic acid to be detected was a blank control, no fluorescent signal value was generated; when the target nucleic acid to be detected was one or more of the HPV types, the corresponding fluorescent signal was collected and detected. This shows that the nucleic acid detection method of the present invention can achieve quadruple detection of a single tube.
  • the result is shown in FIG. 7 .
  • the results show that the detection system can be used for actual clinical detection, and has high sensitivity, high specificity, and rapid effect.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
US17/045,393 2018-04-03 2018-10-12 Method for detecting nucleic acid based on prokaryotic argonaute protein and application thereof Pending US20210164024A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CN201810291873.0A CN108796036B (zh) 2018-04-03 2018-04-03 基于原核Argonaute蛋白的核酸检测方法及其应用
CN201810291873.0 2018-04-03
PCT/CN2018/110056 WO2019192156A1 (zh) 2018-04-03 2018-10-12 基于原核Argonaute蛋白的核酸检测方法及其应用

Publications (1)

Publication Number Publication Date
US20210164024A1 true US20210164024A1 (en) 2021-06-03

Family

ID=64095525

Family Applications (1)

Application Number Title Priority Date Filing Date
US17/045,393 Pending US20210164024A1 (en) 2018-04-03 2018-10-12 Method for detecting nucleic acid based on prokaryotic argonaute protein and application thereof

Country Status (5)

Country Link
US (1) US20210164024A1 (zh)
EP (1) EP3792364A4 (zh)
JP (1) JP7224676B2 (zh)
CN (1) CN108796036B (zh)
WO (1) WO2019192156A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114561374A (zh) * 2022-03-11 2022-05-31 上海交通大学 一种新型嗜热核酸内切酶突变体及其制备方法和应用
CN116103419A (zh) * 2023-03-07 2023-05-12 天津科技大学 一种基于Argonaute的通用型一步法检测食源性致病菌的方法及应用

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109880891B (zh) * 2019-04-22 2021-07-30 上海交通大学 基于核酸酶偶联pcr原理富集低丰度dna突变的检测技术体系及应用
CN110283941A (zh) * 2019-06-28 2019-09-27 湖北大学 一种用于hpv分型检测的试剂盒与方法
CN110331202A (zh) * 2019-07-29 2019-10-15 湖北大学 一种用于检测brac1基因snp的试剂盒与方法
CN112029903B (zh) * 2020-08-13 2021-11-16 湖北大学 一种SARS-CoV-2病毒核酸检测的引物组、试剂盒及其应用
CN111926117B (zh) * 2020-08-18 2022-09-13 交弘生物科技(上海)有限公司 SARS-CoV-2病毒核酸等温快速检测试剂盒及检测方法
CN112538470B (zh) * 2020-12-11 2023-03-28 湖北大学 一种原核生物来源的Argonaute蛋白及其应用
CN113046355B (zh) * 2021-04-20 2023-04-07 上海交通大学 一种中温原核Argonaute蛋白PbAgo表征及应用
CN113106087B (zh) * 2021-04-20 2023-02-24 上海交通大学 一种新型高温Argonaute蛋白的表征及应用
CN113201578B (zh) * 2021-04-29 2023-06-23 上海交通大学 一种新型高温Argonaute蛋白TpsAgo表征及应用
CN114277109B (zh) * 2021-10-21 2023-12-26 上海交通大学 基于常温原核Argonaute蛋白的核酸检测方法及其应用
CN114085892B (zh) * 2021-11-30 2023-07-28 上海交通大学 用于检测靶标核酸分子的可视化检测体系、试剂或试剂盒及检测方法
CN114606215B (zh) * 2022-01-24 2022-11-29 湖北大学 一种真核生物来源的Argonaute蛋白及其应用
CN114703328B (zh) * 2022-04-25 2023-12-19 湖北大学 PfAgo蛋白介导的B19病毒核酸检测试剂盒及检测方法
CN115896351A (zh) * 2022-11-17 2023-04-04 江苏海洋大学 一种新冠病毒s蛋白l452r突变的基因分型检测方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10253311B2 (en) * 2014-04-10 2019-04-09 The Regents Of The University Of California Methods and compositions for using argonaute to modify a single stranded target nucleic acid

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7482118B2 (en) * 2001-11-15 2009-01-27 Third Wave Technologies, Inc. Endonuclease-substrate complexes
US9902973B2 (en) * 2013-04-11 2018-02-27 Caribou Biosciences, Inc. Methods of modifying a target nucleic acid with an argonaute
WO2016161375A2 (en) * 2015-04-03 2016-10-06 University Of Massachusetts Methods of using oligonucleotide-guided argonaute proteins
EP3303585A4 (en) * 2015-06-03 2018-10-31 Board of Regents of the University of Nebraska Dna editing using single-stranded dna
CN107513538A (zh) * 2016-06-17 2017-12-26 北京大学 基因敲除方法
EP3455357A1 (en) * 2016-06-17 2019-03-20 The Broad Institute Inc. Type vi crispr orthologs and systems
CN106555011B (zh) * 2016-07-18 2023-12-19 德诺杰亿（北京）生物科技有限公司 基因检测或基因分型的组合物及方法
CN107805634A (zh) * 2016-09-07 2018-03-16 北京大学 用于序列操纵的Argonaute‑核酸序列组分系统、方法以及组合物
CN106589134B (zh) * 2016-11-11 2021-04-20 仪宏 嵌合蛋白pAgoE及构建方法、应用以及使用向导的嵌合蛋白pAgoE及构建方法、应用
CN107488710B (zh) * 2017-07-14 2020-09-22 上海吐露港生物科技有限公司 一种Cas蛋白的用途及靶标核酸分子的检测方法和试剂盒

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10253311B2 (en) * 2014-04-10 2019-04-09 The Regents Of The University Of California Methods and compositions for using argonaute to modify a single stranded target nucleic acid

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Enghiad et al. (Synthetic Biology, 2017, p. 752-757) *
Wierer et al. (PLOS ONE, p. 1-19, May 2016) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114561374A (zh) * 2022-03-11 2022-05-31 上海交通大学 一种新型嗜热核酸内切酶突变体及其制备方法和应用
CN116103419A (zh) * 2023-03-07 2023-05-12 天津科技大学 一种基于Argonaute的通用型一步法检测食源性致病菌的方法及应用

Also Published As

Publication number Publication date
CN108796036A (zh) 2018-11-13
JP7224676B2 (ja) 2023-02-20
JP2021517469A (ja) 2021-07-26
EP3792364A1 (en) 2021-03-17
EP3792364A4 (en) 2021-08-18
WO2019192156A1 (zh) 2019-10-10
CN108796036B (zh) 2021-11-19

Similar Documents

Publication Publication Date Title
US20210164024A1 (en) Method for detecting nucleic acid based on prokaryotic argonaute protein and application thereof
US11041189B2 (en) Genetic markers for discrimination and detection of a virus causing red sea bream iridovirus disease which is an infectious aquatic organism disease, and method of discriminating and detecting the virus using the same
JP6177844B2 (ja) 試験サンプル中のヒトパピローマウイルスおよびヒトベータグロビン配列を検出するためのプライマーおよびプローブ
CN110964814B (zh) 用于核酸序列变异检测的引物、组合物及方法
CN110117675B (zh) 一种实时荧光定量pcr检测rcl的试剂和方法
CN114107574A (zh) 检测新型冠状病毒及其Omicron突变株的试剂盒和方法
CN111534514A (zh) 一种基于Crisper的新型冠状病毒的检测试剂盒
CN111926114A (zh) 一种检测副流感病毒多重real-time PCR试剂盒、方法和应用
CN114410844A (zh) 多重qPCR检测GALV和ALB拷贝数判定CAR-T细胞产品中RCR阴阳性的方法
CN112111609A (zh) 一种肠道病毒通用型核酸检测试剂盒
CN105838826B (zh) 一种快速区分犬细小病毒疫苗株与野毒株的双色荧光pcr引物、探针及方法
CN116814859A (zh) 鉴别非洲猪瘟病毒基因ⅰ型和ⅱ型的引物探针组合物、试剂盒及方法
CN116479177A (zh) 一种用于新型冠状病毒s基因6种突变位点检测的引物探针组合
CN112322784B (zh) 寡聚核苷酸组、试剂盒及其应用
CN116064954A (zh) 一种基于mb-rt-pcr检测新型冠状病毒及分型的试剂盒及其应用
KR102435116B1 (ko) CRISPR-Cas 시스템과 멀티플렉스 LAMP 프라이머 세트를 이용한 인플루엔자 바이러스의 검출 방법
CN110616261A (zh) 一种用于检测egfr基因t790m突变的试剂盒及检测方法
CN112899385A (zh) 鉴别布鲁氏菌s2疫苗株与野毒株的引物组、探针及其应用
KR102529399B1 (ko) 실시간 중합효소연쇄반응을 이용한 수두바이러스 검출 및 정량 방법
CN116694816A (zh) SARS-CoV-2、甲型流感病毒和乙型流感病毒核酸等温多重检测试剂及检测方法
Karić Polymerase Chain Reaction [PCR] methods
CN115851886A (zh) 一种基于LAMP扩增的荧光检测与CRISPR/Cas检测的双重检测体系及应用
CN116103443A (zh) 用于prrsv谱系1c变异株crispr检测的引物组、试剂盒及其检测方法
CN116949214A (zh) 一种猫冠状病毒和猫细小病毒检测引物探针组合物、试剂盒和检测方法
CN116200545A (zh) 一种检测3种病毒的Taqman荧光定量PCR试剂盒及方法

Legal Events

Date Code Title Description
AS Assignment

Owner name: SHANGHAI JIAO TONG UNIVERSITY, CHINA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FENG, YAN;XUN, GUANHUA;LIU, QIAN;AND OTHERS;SIGNING DATES FROM 20201013 TO 20201015;REEL/FRAME:054521/0368

STPP Information on status: patent application and granting procedure in general

Free format text: APPLICATION DISPATCHED FROM PREEXAM, NOT YET DOCKETED

AS Assignment

Owner name: FENG, YAN, CHINA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SHANGHAI JIAO TONG UNIVERSITY;REEL/FRAME:057017/0795

Effective date: 20210715

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

AS Assignment

Owner name: JIAOHONG BIOTECHNOLOGY (SHANGHAI) CO., LTD., CHINA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:FENG, YAN;REEL/FRAME:057538/0100

Effective date: 20210915

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER